1. Field of the Invention
A process is provided wherein hemoglobin, a fragile material, is formulated into high hemoglobin content aqueous hemoglobin solution in oil in water multiple emulsions while maintaining high oxygen exchange activity necessary for the below uses. Hemoglobin multiple emulsion having specified properties is suitable for provision of oxygen as a blood substitute and other oxygen transfer processes. A double emulsion of aqueous high hemoglobin content in physiologically compatible mineral oil or fixed oil in an outer aqueous saline solution is provided in sufficiently small droplet size to provide oxygen flow through blood vessels to desired body tissues or organs.
2. Description of Relevant Art
It is important in many physiological and industrial applications to have available an oxygen carrying chemical for provision of oxygen to an oxygen depleted environment. One of the most important applications is provision of an effective oxygen carrying blood substitute. In addition to emergency situations where there are not adequate supplies of whole blood, there are advantages in use of a synthetic blood substitute over the use of whole blood. For example, the efficiency of oxygenation by deficient blood flow in a tissue or an organ resulting from restriction of a blood vessel cannot be treated by use of whole blood, whereas blood substitutes of low bulk viscosity may deliver oxygen through constricted vessels, thereby preventing heart attacks and strokes caused by constriction of the arteries. Use of synthetic blood substitutes also eliminates transmission of blood borne infectious diseases, such as hepatitis and acquired immune deficiency syndrome. Other problems of intolerance or allergy to blood may be solved by synthetic blood substitutes.
An ideal synthetic blood substitute should have high oxygen carrying capacity and low oxygen affinity to permit loading of oxygen in the lungs and releasing of oxygen in the tissue; colloid osmotic pressure close to that of blood plasma; viscosity the same or less than that of whole blood; non-toxicity to the human body; histocompatibility, no antigenic affects; an adequate lifetime in the circulatory system to meet the desired needs for oxygen provision; relatively rapid metabolism or excretion of chemical agents; and adequate storage stability. To date, no blood substitutes have been fully approved for use in the United States of America.
One approach to provision of blood substitutes has been use of media with high passive oxygen solubility, primarily perfluorocarbon emulsions, which have been found to be unstable, have inadequate oxygen carrying capacity, and are toxic to the human. Problems with many perfluorocarbon emulsions have been high oxygen concentrations necessary due to the fluorocarbon emulsion carrying oxygen by passive solubility and the necessity to store the emulsion in the frozen state to retain stability.
The most promising present approaches involve use of chemical hemoglobin in various forms. Although stroma-free hemoglobin solutions have an adequate oxygen carrying capacity, they have high oxygen affinity, high colloid osmotic pressure, possible toxicity, and clearance from the cardiovascular circulation which is too rapid. One problem with stroma-free hemoglobin solutions has been that their oxygen affinity is much higher than that of normal hemoglobin in red blood cells and therefore oxygen is preferentially extracted from the cellular hemoglobin. Sehgal, L. R., Gould, S. A., Rosen, A. L., Moss, G. S.: Appraisal of Red Cell Substitutes: Hemoglobin Solution and Perfluorochemical Emulsions, Laboratory Medicine, 14:545, 1983; Gould, S. A., Rosen, A. L., Sehgal, L. R., Moss, G. S.: Red Cell Substitutes: Hemoglobin Solution or Fluorocarbon?, J. Trauma, 22:736, 1982; Gould, S. A., Rosen, A. L., Sehgal, L. R., Moss, G. S.: Hemoglobin Solutions as Red Cell Substitutes; Trans. Am. Soc. Art. Int. Organs, 26:350, 1980. Pyridoxylation followed by polymerization of stromo-free hemoglobin solutions has reduced many of the above problems except for high oxygen affinity and possibly toxicity. Also, the process generates some methemoglobin, which is a form of hemoglobin which cannot transfer oxygen. Sehgal, L. R., Rosen, A. L., Gould, S. A., Moss, G. S.: Polymerized Pyridoxylated Hemoglobin: A Red Cell Substitute with Normal Oxygen Capacity, Surgery, 95:433, 1984: Keipert, P. E., Chang, T.M.S.: Preparation and In-vitro Characteristics of Pyridoxylated Polyhemoglobin as Blood Substitutes, Appl. Biochem. Biotechnol. 10:133, 1984.
Encapsulation of hemoglobin solution in a synthetic cell has been attempted by encapsulating hemoglobin solution within nylon membranes, cross-linked protein membranes, polyhemoglobin membranes and liposomes encapsulating hemoglobin in phospholipid vesicles. Miller, I., Synthetic Blood Substitutes: Where Are We and Where Do We Go From Here?, CRC, Crit. Rev. BioEng., 149-178, Dec. 1978. Hemoglobin solution droplet encapsulation in a polymerized hemoglobin encapsulating membrane using glutaraldehyde as a crosslinking agent is described in "Artificial Red Cells with Crosslinked Hemoglobin Membranes, Thomas A. Davis, William J. Asher and Herbert W. Wallace, Applied Biochemistry and Biotechnology, Vol. 10, pgs. 123-132 (1984). The liposome encapsulated hemoglobin, although overcoming many of the problems encountered with other blood substitute products, are still too rapidly cleared from the circulatory system, are limited in oxygen carrying capacity, and have low encapsulation efficiencies, in the order of 10 to 20 percent. A method of scaled-up production of liposome-encapsulated hemoglobin described in U.S. Pat. No. 4,776,991 overcomes some of the problems pointed out above.
Preparation of multiple emulsions of water in oil in water using non-ionic emulsifiers, deionized distilled water and liquid paraffin with mixing to form the water in oil emulsion and homogenizing to form the oil in water emulsion is taught by "An Attempt at Preparing Water-in-Oil-in-Water Multiple Phase Emulsions", Sachio Matsumoto, Yashiko Kita and Daizo Yonezawa, Journal of Colloid and Interface Science, Vol. 57, No. 2, pgs. 353-361 (1976). Water in olive oil in water emulsions were prepared using a mixed soy lecithin and Span 80 emulsifier which interact to form a viscoelastic film at the oil/water interface and sucrose-fatty acid ester at the outer water phase is taught by "Preparation of Water-in-Olive Oil Multiple-Phase Emulsions in an Eatable Form", Sachio Matsumoto, Yoshiro Ueda, Yoshiko Kito, and Daizo Yonezawa, Agric. Biol. Chem., 42, No. 4, pgs. 739-743 (1978).